Fig. 4
Pitavastatin and atorvastatin affect intrinsic 5-FU resistance in CRC cells. (A-C) Colony formation assay of DLD-1 and DLD-1R cells following treatment with 5-FU, pitavastatin, or atorvastatin for nine days. (D) The Cyto3D™ dead-live assay following cotreatment with 5-FU and pitavastatin or atorvastatin in DLD-1 spheroids. Live-dead imaging of DLD-1 spheroids was conducted using the Cyto3D Live-Dead assay. Bright-field and fluorescence images were acquired using an Olympus IX83 inverted microscope with a 10X objective. The scale bar represents 500 μm. (E-F) DLD-1 and DLD-1R were seeded onto R3CE plates at 1000 cells per well density. 5-FU, pitavastatin, and atorvastatin were added to the culture medium at the indicated concentrations for 7 days. Images were acquired using an Olympus IX83 inverted microscope with a 10X objective. The scale bar represents 500 μm. (G-I) Effects of 5-FU, pitavastatin, and atorvastatin on spheroid formation by DLD-1 and DLD-1R cells. (J-K) Drug-treated DLD-1 and DLD-1R spheroids were stained with CD44 (labeled with Alexa Fluor™ 488), Alexa Fluor™ 568 Phalloidin, and/or Hoechst 33,342. IF staining images were acquired using a Zeiss LSM900 confocal microscope with a 20X objective. The scale bar represents 100 μm. Data represent mean ± SEM (N = 3). DLD-1R: DLD-1 5-FU resistance